Device for selectively filtering under reduced pressure and for vacuum drying sample liquids or drops of sample liquids as well as use of said device

ABSTRACT

A device for selectively filtering liquid samples in compartments comprising inlet and outlet openings of a carrier body under reduced pressure and vacuum drying of the sample liquid in the area of the outlet openings of the compartments. The device comprises a vacuum pump for producing two levels of subpressure, a suction conduit, and a chamber comprising an interior space limited walls by a top wall having an opening with an edge, a bottom wall, and lateral walls connecting the top and bottom walls. A lid or the carrier body with outlet openings directed towards the interior space of the chamber can be placed in a substantially gastight manner on the opening with an edge. One of the lateral walls is formed as an access wall which is opened and substantially gastightly sealed or introducing the carrier body into the interior space of the chamber.

This application is a divisional of co-pending application Ser. No.09/424,537, filed on Nov. 24, 1999, now U.S. Pat. No. 6,315,902 and forwhich priority is claimed under 35 U.S.C. § 120. application Ser. No.09/424,537 is the national phase of PCT International Application No.PCT/EP98/03130 filed on May 27, 1998 under 35 U.S.C. § 371. The entirecontents of each of the above identified applications are herebyincorporated by reference. This application also claims priority ofApplication No. 197 22 02 1.5 filed in Germany on May 27, 1997 under 35U.S.C. § 119.

The invention relates to a device for selectively filtering liquidsamples in compartments comprising inlet and outlet openings of acarrier body under reduced pressure and vacuum drying drops present inthe area of the outlet openings of the compartments. Furthermore, theinvention relates to the uses of the device in the purifying ofbiopolymers, especially nucleic acids.

In molecular biology, in the analysis of samples, carrier bodies calledmicrotitration plates are used comprising a plurality of compartmentsarranged two-dimensionally in lines and rows. These compartments areprovided with inlet openings towards the upper side of the carrier bodyand comprise outlet openings on the lower side of the carrier body. Inthe compartments, there are filter bodies in the shape of frits ormembranes provided to bond the substances of the liquid samples to beanalyzed.

The sample liquids to be analyzed are introduced into the individualcompartments of a carrier body (microtitration plates), if requiredafter being processed. By applying a low pressure to the outlet openingsof the compartments, it is provided that the sample liquids flow throughthe filter bodies of the compartments. In this process, the componentsto be analyzed and other components of the sample liquid get caught inthe filter bodies. In order to obtain the components of interest of theliquid sample, the components which are not required have to be washedout of the filter bodies. For this purpose, wash buffers of variousconcentrations are pipetted into the compartments. By means of thesewash buffers, a part of the components not of interest of the sampleliquids is released form the filter bodies. By means of an elutionbuffer, the component of interest of the sample liquids is then washedout of the filter bodies. The pipetting and the flowing process of thewash buffers, the elution buffer and the sample liquids occur in apipetting machine, which is computer controlled. These pipettingmachines are provided with a vacuum chamber comprising an openingwhereon the carrier bodies are substantially gastightly fitted to drawout the sample liquids or the wash buffers through the filter body. Adevice of the type described above is described in DE-A-41 07 262. Inthe filtration under reduced pressure, it always occurs that remainingliquids stay on the outlet openings of the compartments in the form ofdrops. Before the step of elution takes place, it has to be providedthat these liquid drops have been removed. For the quantities to beanalyzed occasionally are rather small so that a mixture with remainingliquid can lead to faulty results. What is more, components of thesample liquid which are not to be analyzed are present in the liquiddrops, thus also adulterating the result of the analysis. In prior art,the carrier bodies, respectively microtitration plates, are droppedseveral times on a support having an absorbent material so that theliquid drops are virtually shaken off. However, this process cannot beintegrated in the case of an automatic process, as in the case of apipetting machine.

The invention is based on the objective to provide a device by means ofwhich remaining liquid drops on the outlet opening of the compartmentsof the carrier bodies can be removed automatically and without manualinteraction after a filtration process under reduced pressure has takenplace.

To achieve this objective, the invention proposes a device forselectively filtering liquid samples in compartments comprising inletand outlet openings of a carrier body under reduced pressure and dryingdrops of the sample liquid present in the area of the outlet openings ofthe compartments, the device comprising:

-   -   a chamber comprising an interior space limited by a top wall, a        bottom wall and lateral walls connecting them,    -   the top wall comprising an opening with an opening edge whereon        a closing lid or the carrier body can selectively be put in a        substantially gastight manner by means of outlet openings        directed towards the interior space of the chamber,    -   one of the lateral walls being formed as an access lateral wall        which can be opened and closed substantially gastightly for        introducing the carrier body into the interior space of the        chamber, and    -   the bottom wall comprising a drain for liquid exiting from the        outlet openings of the compartments of the carrier body, and    -   a vacuum pump with a suction conduit terminating in the interior        space of the chamber for producing a first low pressure for        drawing liquid samples through the outlet openings of the        compartments with the carrier body being placed on the opening        edge of the top wall and the access lateral wall of the chamber        being closed and for producing a second low pressure higher than        the first low pressure for drying drops of liquid samples still        present on the outlet openings of the compartments with the        carrier body being located in the interior space of the chamber        and the lid being placed on the opening edge of the top wall as        well as the access lateral wall of the chamber being closed.

The device according to the invention is provided with a low pressurechamber comprising a top wall, a bottom wall and lateral wallsconnecting them. One of the lateral walls is formed as an access lateralwall and can be opened and closed so that it is possible to introduce acompartment carrier body (subsequently called microtitration plate) viathis lateral wall into the interior space of the chamber. Furthermore,the chamber comprises an opening in the top wall thereof being closableby means of a lid. As an alternative to the lid, a microtitration platecan also be placed onto the opening so that the chamber can then be usedfor filtering the sample liquid of the compartments under reducedpressure. The chamber comprises a drain in the bottom wall so that theflowing liquids can be drained.

The low pressure in the interior space of the chamber is produced by avacuum pump connected to the interior space of the chamber by means of asuction conduit. By means of this vacuum pump, a first and a second lowpressure can be produced alternatively in the interior space of thechamber. The first low pressure is required to draw in the sample liquidthrough the outlet opening of the compartments of the microtitrationplate. Thus, the first low pressure is used for filtering under reducedpressure. The second low pressure higher than the first low pressureserves to dry sample liquid drops which might be adhering to the outletopenings of the microtitration plate at a vacuum. In the case of vacuumdrying, the opening in the top wall of the chamber is closed by means ofa lid, while the microtitration plate itself is located in the interiorof the chamber. By lowering the pressure in the chamber, the evaporationpoint of the liquid drops is reduced, whereby they can be removed byevaporation.

By means of the device according to the invention, it is possible to useone and the same low pressure chamber both for filtering and forremoving sample liquid drops. The manipulations to be executed on thechamber for this purpose, which are described above, can be put intopractice without a problem by means of the gripping device of apipetting machine so that manual interference is no longer necessary.

In an advantageous development of the invention, it is provided that thechamber comprises a support mounting for receiving the microtitrationplate for putting it in the interior space of the chamber. Preferably,this support mounting is located on the interior side of the accesslateral wall of the chamber directed towards the interior space, whichwall is formed to be movable from the other walls of the chamber ortowards them for this purpose. In this context, it is advantageous ifthe access lateral wall is provided with at least one cam elementconnected to the access lateral wall. The cam element can be moved bymeans of a driving device, whereby the access lateral wall is alsomoved. In this context, the cam element and the access lateral wall workin the manner of an automatic drawer which can be opened so far that themicrotitration plate can be inserted from the top into the receptionmounting by means of a gripping arm and can be taken out of the supportmounting from the top, respectively.

In an advantageous development of the invention, it is further providedthat the chamber comprises at least one heating element for heating theinterior space. By means of this heating element, an appropriatetemperature level can be set in the interior space of the chamber. Theinterior space of the chamber can also be heated during filtering underreduced pressure. In any case, heating the interior space isadvantageous in the case of vacuum drying, as the conditions in thechamber can then be set more quickly such that liquid drops which mightstill be present are evaporated.

In order to simplify placing the lid or the microtitration plate on theopening edge of the top wall, it is suitable if the opening edge isprovided with introduction surfaces extending angularly to the outsidefor centering the lid or the microtitration plate when it is put on.This has the advantage that the opening of the top wall can be closedautomatically either by means of the lid or the microtitration plate,even if the positioning of the gripping arm is less accurate.

In order to be able to seal the interior space of the chamber againstthe environment without having moved the microtitration plate into theinterior space of the chamber before and having closed the opening inthe top wall by means of the lid, it is advantageous to insert a sealingbody or a sealing mat consisting of a substantially gastight materialand being placed on top of the inlet opening of the compartments of themicrotitration plate, if it is located in the opening of the top wall.After a filtration under reduced pressure has taken place, themicrotitration plate can remain in the opening of the top wall to forvacuum drying liquid drops which might be adhering to the outletopenings.

The device according to the invention can especially be employed forisolating, separating and/or purifying of biopolymers, as is describedin EP-A-0268946; this document is incorporated by reference in thedisclosure of the application.

An exemplary embodiment of the invention will now be described withreference to the figures.

FIG. 1 shows a lateral view of a pipetting machine with a gripping armand a low pressure chamber,

FIG. 2 shows a longitudinal section through the low pressure chamber inthe closed state and with the carrier body being placed on top,

FIG. 3 shows a section along line III—III of FIG. 2,

FIG. 4 shows a longitudinal section through the low pressure chamber inthe opened state to show the introduction of the carrier body into thelow pressure chamber, and

FIG. 5 shows a longitudinal section through the low pressure chamber inthe closed state with the opening in the top wall being closed and thecarrier body being located in the interior of the low pressure chamberfor vacuum drying.

FIG. 1 shows, strongly schematized, a pipetting robot 10 comprising amovable bracket 12. The bracket 12 is movable along the rail 14 andcomprises one or multiple pipetting needles 16 as well as a grippingdevice 18 with two gripping arms 20. The pipetting needles 16 and thegripping device 18 are movable along the bracket 12, thus being able tomove in the X and Y directions relative to the table 22 of the machine10.

Various utensils and objects required for the pipetting process arelocated on the table 22, which are not elaborated on for reasons ofsimplicity. Among other things, a low pressure chamber 24 is located onthe table 22 which can be employed both for filtering under reducedpressure and for vacuum drying remaining liquid which might still beadhering to the carrier bodies 26 after a filtering process.

The construction of the low pressure chamber 24 and the carrier body 26will subsequently be described with reference to FIGS. 1 to 5. As can beseen in FIG. 2, the carrier body 26 comprises a plurality of tubularcompartments 28 each comprising an inlet opening 30 and an outletopening 32. Filter bodies 34 in the form of porous matrix materials,frits or membranes are located in the compartments 28. The carrier body26 comprises a rim 36 extending circumferentially laterally over thearrangement of the compartments 28.

The chamber 24 comprises a bottom wall 38 and a top wall 40 parallelthereto which are interconnected via four lateral walls 42, 44, threelateral walls of which (designated by 42 in the figures) are formed tobe fixed and the fourth lateral wall 44 is formed movably to enableaccess to the interior space 46 of the chamber 24 (subsequently calledaccess lateral wall). An opening 48 is located in the top wall 40, theopening edge 50 thereof comprising a sealing surface 52. Above thesealing surface 52, the opening edge 50 comprises an expanding centeringzone 54.

The access lateral wall 44 is provided with a support mounting 56comprising a circumferential frame 58 defining an insertion opening 60and being fixedly connected from the inside to the access lateral wall44. The frame 58 is arranged parallelly to the top wall 40 and thebottom wall 38. As can be seen especially in the case of FIGS. 3 and 4,two cam elements 62 project from the access lateral wall 44 which areguided axially displaceably in guiding rails 64 located in a base plate66 supporting the bottom wall 38. A driving motor 68 is flanged on theplate 66 comprising a driving element 70 in the form of a cog wheelcombing with a rack 72 connected to one of the two cam elements 62. Inthis manner, the access lateral wall 44 can be moved in the direction ofthe arrows 73 by means of the fixed lateral walls 42 or can be movedtowards them. On the interior side of the access lateral wall 44, acircumferential seal 74 is located abutting on the fixed lateral walls42, the top and the bottom walls 40, 38 and seals the interior space 46of the chamber 24.

As can further be seen in the figures, the bottom wall 38 comprises aninclination 76 towards a drain pipe 78 to direct the liquid to areception container 80. A vacuum pump 82 is connected to the drain pipe78 via a suction conduit 81, through which pump air is drawn out of theinterior space 46 to produce a low pressure. As can be seen in FIG. 5,the opening 48 in the top wall 40 of the chamber 24 can be closed bymeans of a lid 84 comprising a projection 86 for gripping the lid 84 bymeans of the gripping device 18 of the pipetting machine 10. Apart fromthe pressure, the temperature 46 of the chamber 24 can also be set, withat least one of the fixed lateral walls 42 being provided with one ormultiple heating elements 87.

Subsequently, the operation and the use of the chamber 24 by means ofthe pipetting machine 10 will be elaborated on.

For the liquids present in the compartments 28 of the carrier body 26 topass through the filter bodies 34, the carrier body 26 is inserted intothe opening 48 of the top wall 40 of the chamber 24, the projecting rim36 being placed on the sealing surface 52. The carrier body 26 isinserted into the opening 48 by means of the gripping device 18, withthe carrier body 26 being centered in the opening 48 by the inlet zone54 of the opening edge 50. During this procedure, the access lateralwall 44 is closed, with no carrier body 26 being located in the insertopening 60 of the support mounting frame 58. Then a first low pressureis produced in the interior space 46 of the chamber 24 due to which theliquids flow through the filter bodies 34. In this process, the lowpressure amounts to about 400 to 800 mbar. During this phase, theheating elements 87 can be operated to pre-heat the interior space 46 ofthe chamber 24. The liquid flowing out of the outlet openings 32 of thecompartments 28 gets into the reception container 80 via the drain pipe78. The liquid can therefore be disposed of by other means, as theresidues remaining in the filter bodies 34 are of interest for theanalysis.

To make these residues available for analysis, they have to be releasedfrom the filters 34. For this purpose, the carrier body 26 has to beplaced onto a mounting 88 with a plurality of individual receptioncontainers 90 in which the substances being released from the individualfilter bodies 34 are received (see FIG. 1). As the quantities of thesubstances to be analyzed, respectively the components of the liquids,are rather small, and for the analysis to be performed correctly, it hasto be provided that only the components to be analyzed get into thereception containers 90. Liquid drops which might still be adhering tothe outlet openings 32 from the preceding filtering step can adulteratethe measuring results, which is why they have to be removed before theelution process. For this purpose, the carrier body 26 located in theopening 48 of the top wall 40 is elevated by means of the grippingdevice 18 to introduce the carrier body 26 from the top into theinsertion opening 60 of the frame 58. For this purpose, the motor 68 isactuated to move out the access lateral wall 44 until the frame 58 iscompletely moved out of the interior space 46 of the chamber 24. Thissituation is shown in FIG. 4, by means of which it becomes clear how thecarrier body 26 is now inserted in the insertion opening 60 from thetop, the edge thereof, like the opening edge 50, comprising an inletzone 92. In this process, the control of the gripping device 18 can beselected such that the carrier body 26 is dropped shortly after beingelevated and moved out of the opening 48 so that liquid drops whichmight still be adhering to the outlet openings 32 are released by thevibrations resulting therefrom. After the insertion of the carrier body26 into the frame 58, the access lateral wall 44 is moved back towardsthe fixed lateral walls 42. Additionally, the gripping device 18 gripsthe lid 84 to close the opening in the top wall 40 with it.

Then the interior space 46 now substantially gastightly sealed isexposed to a low pressure which is substantially greater than the lowpressure to draw the liquid through the filter body 34. The low pressurenow establishing amounts to about 50 to 100 mbar. Additionally, atemperature range between 40 and 60° C. is set by correspondinglycontrolling the heating elements 87 in the interior space 46. That way,the evaporation point of the liquid which might still be adhering to theoutlet openings 32 is shifted, the liquid thus evaporating. After thisvacuum drying process, the chamber 24 is re-aerated, and the accesslateral wall 44 is moved so that the carrier body 26 liberated fromliquid drops on the outlet openings 32 can be moved onto the mounting 88for applying the elution buffer. Then the carrier body 26 is moved ontothe opening 48 of the top wall 40 of the chamber 24. A carrier body withcompartments is inserted into the frame 58, in which compartments theeluate of the compartments of the carrier body 26 is received, when itis drawn out therefrom by means of low pressure.

1. A method for selectively filtering mixtures containing biopolymersutilizing an apparatus containing compartments comprising inlet andoutlet openings of a carrier body under reduced pressure and for vacuumdrying drops of the mixtures present in the area of the outlet openingsof the compartments, comprising: a chamber comprising an interior spacelimited by a top wall, a bottom wall and lateral walls connecting them,the top wall comprising an opening with an opening edge whereon aclosing lid or the carrier body can selectively be placed in asubstantially gastight manner by means of outlet openings directedtowards the interior space of the chamber, one of the lateral wallsbeing formed as an access lateral wall which can be opened and closedsubstantially gastightly for introducing the carrier body into theinterior space of the chamber, and the bottom wall comprising a drainfor liquid exiting from the outlet openings of the compartments of thecarrier body, and a vacuum pump with a suction conduit terminating inthe interior space of the chamber for producing a first low pressure fordrawing liquid samples through the outlet openings of the compartmentswith the carrier body being placed on the opening edge of the top walland the access lateral wall of the chamber being closed and forproducing a second low pressure higher than the first low pressure fordrying drops of liquid samples still present on the outlet openings ofthe compartments with the carrier body being located in the interiorspace of the chamber and the lid being placed on the opening edge of thetop wall as well as the access lateral wall of the chamber being closed,said method comprising separating, isolating and/or purifyingbiopolymers from said mixtures containing said biopolymers.
 2. Themethod according to claim 1, wherein the biopolymers comprise peptides,proteins, nucleic acids, especially DNA and/or RNA, oligo- and/orpolysaccharides.
 3. The method according to claim 1, wherein the DNA isa genomic or a plasmid DNA.
 4. A method for isolating a substance ofinterest, especially a biopolymer, from a sample comprising differentsubstances and being located in a compartment having an inlet opening aswell as an outlet opening and a porous matrix material arrangedtherebetween, to which the substances of the sample are immobilized bymeans of different affinities, comprising the following steps: feeding awash buffer into the inlet opening of the compartment for releasingthose substances from the matrix material the affinity of which to thematrix material is smaller than the affinity by means of which thesubstance of interest is bound to the material, drawing the wash bufferand the solved substances through the matrix material by means of lowpressure and out of the outlet opening of the compartment, by using thedevice operated for filtering under reduced pressure according to claim1, removing of residual material which might still be present on theoutlet opening of the compartment, and feeding an elution buffer intothe inlet opening of the compartment for releasing substantially merelythe substance of interest from the matrix material, drawing the elutionbuffer and the substance of interest by means of low pressure throughthe matrix material and out of the outlet opening of the compartment, byusing the device operated for filtering under reduced pressure, andduring filtration, collecting the elution buffer and the substance ofinterest exiting from the outlet opening into a compartment insertedinto the device.
 5. The method according to claim 4, wherein thesubstance of interest is a nucleic acid, oligo- or polynucleotide.